Many small proteins show evidence for conformational changes prior to the rate-limiting step in the formation of the densely packed native structure. Despite intense study, our understanding of the physical properties and mechanistic role of these early folding intermediates remains incomplete. In particular, little is known about the chain topology and tertiary structural preferences in early intermediates, which are critical for understanding how folding is initiated and directed along productive channels. A major goal of this project is to elucidate the structural properties of the transient states and kinetic barriers encountered during early stages of folding of two representative model proteins, protein G and staphylococcal nuclease. Initial stages of folding extending well into the microsecond time scale will be explored by coupling advanced rapid mixing methods with structurally informative conformational probes, such as intrinsic and extrinsic fluorescence probes, and protection of individual amide hydrogens from solvent exchange monitored by NMR. The involvement of specific residues and interactions in stabilizing transient states and barriers in folding of protein G will be explored by combining these kinetic methods with site-directed mutagenesis. The results will identify key structural features involved in each stage of folding of this prototypic single-domain protein. Detailed insight into the formation of hydrophobic clusters, specific fluorescence quenching interactions and long-range distance distributions during folding of staphylococcal nuclease will be obtained by kinetic analysis of variants with engineered tryptophan residues and fluorescence energy transfer methods. Complementary information on the formation of hydrogen bonded structure during early stages of folding will be obtained by combining ultrarapid quenched-flow methods with NMR-detected hydrogen exchange. The insight into fast folding events for these and other proteins with diverse structural properties will provide a firm experimental basis for testing theoretical and computational models of protein folding, structure prediction and de novo protein design.